IgA nephropathy (IgAN) is the most common primary glomerulonephritis and an important cause of end-stage kidney failure. It is a mesangioproliferative glomerulonephritis defined by IgA1 mesangial deposits. Although it has been speculated for some time that the pathogenesis of IgAN is driven by deposition of circulating immune complexes (IC), this has been difficult to prove due to the lack of animal models of IgAN. Our development of new protocols that permit formation of engineered IC in vitro and establishment of a passive mouse model of IgAN provide an unprecedented opportunity to elucidate the pathophysiology of IgAN and identify potential therapeutic targets. In IgAN, a fraction of circulating IgA1 has galactose-deficient O-glycans (Gd-IgA1) and is present in circulating IC bound by glycan-specific autoantibodies. We now have characterized Gd-IgA1 and the anti-Gd-IgA1 autoantibodies that are present in these IC and used targeted proteomic approaches to both define the serum factors that associate with these IC and may contribute to their pathogenic effects and to identify the mesangial-cell receptors for the ICs. Rational extension of these studies required analysis of the molecular effects of these pathogenic IgAN ICs on the signaling events that lead to mesangial-cell activation. Global kinase-activity profilin using an innovative peptide substrate microarray platform of human mesangial cells stimulated with Gd-IgA1 IC identified robust tyrosine kinase activity as a major player in IC-induced signaling in three predominant pathways. Similar results were obtained with native and engineered ICs. The mesangial-cell responses after stimulation with these IC were typical of IgAN but differed from those obtained by using IC lacking all of the components in pathogenic ICs. An association with pathogenesis was demonstrated using protein-kinase inhibitors, which confirmed that one of the inhibitors completely blocked IC- mediated mesangial cell proliferation in vitro as well as in vivo in the passive mouse model of IgAN. These data suggest the hypothesis that Gd-IgA1-containing ICs represent a key hit in the pathogenesis of IgAN by activating mesangial cells through specific signaling pathways; the corollary is that this IC-drive signaling in mesangial cells can be blocked by small-molecular-mass inhibitors of protein kinases and thus represents a feasible therapeutic target(s). The team of basic and clinical investigators that has developed the powerful proteomic, kinomic, and cellular approaches used to generate the preliminary data will now test this hypothesis by: 1) Defining the characteristics of Gd-IgA1-containing ICs from sera of patients with IgAN that activate human mesangial cells; 2) Characterizing the signaling pathways activated by Gd-IgA1-containing ICs in mesangial cells; and 3) Determining the efficacy of small-molecular-mass inhibitors of key protein kinases on mesangial cell activation in vivo using the animal model of IgAN. Relevance: The results will shed light on the pathogenesis of IgAN and identify therapeutic targets for disease-specific treatment of IgAN as well as potential response/prognostic biomarkers.